EP0888386B1 - Stable factor viii/ von willebrand factor complex - Google Patents

Abstract

There are disclosed a stable factor VIII/vWF-complexes, particularly comprising high-molecular vWF multimers, being free from low-molecular vWF molecules and from proteolytic vWF degradation products, as well as methods of producing these complexes.

Description

The invention relates to a stable virus-safe factor VIII complex, the particularly high molecular weight vWF multimers with high contains structural integrity and is free of low molecular weight vWF molecules and proteolytic vWF degradation products. Furthermore The invention relates to methods of extraction and manufacture a stable factor VIII complex as well as pharmaceutical Preparations of it.

Blood clotting is a complex process that is sequential Interaction of a number of components, especially fibrinogen, Factor II, factor V, factor VII, factor VIII, factor IX, Includes factor X, factor XI and factor XII. The loss one of these components or the inhibition of their functionality leads to an increased tendency to clot, which may be life-threatening to some patients.

Von Willebrand factor (vWF) circulates in plasma complexed with factor VIII, whereby factor VIII supports blood clotting and vWF in the complex with factor VIII stabilizes it and protects it from proteolytic degradation. Due to its function in platelet aggregation, vWF also intervenes directly in blood clotting. The vWF is a glycoprotein that is produced in various mammalian cells and then released into the circulation. Starting from a polypeptide chain with a molecular weight of approx. 220 kD, a vWF dimer with a molecular weight of 550 kD is formed in the cells by forming several sulfur bridges. The vWF dimers are then used to link other polymers of the vWF with an ever higher molecular weight, up to 20 million daltons. vWF therefore exists in plasma in a series of multimeric forms with a molecular weight of 1 x 10 ⁶ to 20 x 10 ⁶ daltons. It is believed that the high molecular weight vWF multimers in particular are of essential importance in blood coagulation.

In addition to the carrier function for coagulation factor VIII for the vWF the function in the formation of bridges between the vessel wall and platelet and platelet agglutination. The basis for Platelet agglutination is due to the binding of the vWF to surface receptors (Glycoproteins Ib, IIb / IIIa). The binding site is within the vWF for binding to GP Ib in the disulfide loop Cys (509) -Cys (695). It is known that the Platelet agglutination with binding of the vWF to the glycoprotein Ib begins. Then comes an activation signal for binding the vWF to the glycoprotein IIb / IIIa complex and the Agglutination. The platelet agglutination thus sets the bond of the vWF to the surface receptors; through the bond multiple platelets by a vWF molecule occur in succession for agglutination. vWF platelet binding thus represents the molecular one Cause of platelet agglutination.

In hemophilia, blood clotting is due to a lack of certain plasmatic blood coagulation factors disturbed. In the Hemophilia A, the tendency to bleed is due to a lack of factor VIII or a lack of vWF, which is an essential part of factor VIII. Treatment of hemophilia A is done primarily by replacing the missing coagulation factor by factor concentrates e.g. by infusion of factor VIII, factor VIII complex or vWF.

The vWF syndrome has several clinical pictures that focus on one Under or overproduction of the vWF can be attributed. So leads e.g. an overproduction of vWF to the increased tendency of thrombosis, whereas an insufficient supply due to the absence or a reduction of high molecular forms of the vWF is justified, which manifests itself by the fact that an increased Bleeding tendency and prolonged bleeding time due to an inhibited Platelet aggregation occurs. The lack of vWF can also cause phenotypic haemophilia A because of the vWF is an integral part of functional factor VIII. In in these cases the half-life of factor VIII is reduced so that its function in the blood coagulation cascade is impaired is. Patients with von Willebrand syndrome (vWD) therefore often factor VIII deficiency. In these patients the reduced factor VIII activity is not the consequence a defect of the X chromosomal gene, but is an indirect one Follow the quantitative and qualitative change in vWF in plasma. The distinction between hemophile A and vWD can usually be done by measuring the vWF antigen or by determining of ristocetin cofactor activity. Both the vWF antigen content as well as the ristocetin cofactor activity decreased in most vWD patients, whereas in hemophilia A patient is normal.

Conventional methods for the therapy of von Willebrand syndrome done with plasma-derived vWF, being a series of There are approaches to vWD patients with purified vWF or factor VIII / vWF complex to treat.

Purification of factor VIII or factor VIII complex from plasma or cryoprecipitate is particularly difficult in that Factor VIII occurs only in very small amounts in the plasma, extremely is unstable and the association of factor VIII with vWF under specific conditions is reversible. Factor VIII is affected by Purification and concentration obtained from plasma, however, can it depends on the cleaning process for instability and loss the factor VIIl activity due to the separation of vWF and factor VIII come during cleaning. The end product is therefore often a mixture of stable factor VIII complex and unstable Factor VIII, as well as contaminating proteins, e.g. Fibrinogen, fibronectin, or vitamin K-dependent proteins could not be removed by cleaning. Due to the Instability of the cleaned complex becomes stabilizers, such as albumin or amino acids etc. added. The presence of contaminating proteins and / or stabilizers in the purified However, the product reduced the specific activity of the Factor VIII complex.

EP 0 468 181 describes a method for cleaning factor VIII from human plasma by ion exchange chromatography, Elution of factor VIII with high ionic strength at acidic pH and Collecting the eluate in the presence of a stabilizer such as heparin Albumin and PEG and lysine or histidine as antiprotease. However, the specific activity decreases after the addition of albumin from 300 to 1200 U / mg protein to 18-24 U / mg protein.

Madaras et al. (Haemostasis 7: 321-331 (1978)) describe one Process for the purification of factor VIII on heparin-Sepharose and Elution with increasing NaCl concentrations. The so obtained However, factor VIII had little activity.

US 5,252,709 describes methods for separating factor VIII, vWF, fibronectin and fibrinogen from human plasma, where first factor VIII, vWF and fibronectin to an ion exchanger be bound by DEAE type and then by increasing Salt concentrations are eluted separately from the ion exchanger.

Zimmerman et al. (US 4,361,509) describe a process for the purification of factor VIII, the factor VIII / vWF complex being bound to a monoclonal anti-vWF antibody and factor VIII being dissociated from the complex by CaCl ₂ ions. The factor VIII thus obtained is then obtained in a pure form via a further chromatography step, but must be stabilized by adding human albumin.

Through the expression of factor VIII in recombinant cells (Wood et al. (1984), Nature 312: 330-337) was able to genetically factor VIII can be produced, however, only by adding or co-expression of vWF a commercially usable yield on recombinant factor VIII can be achieved. For the production of a pharmaceutical preparation is however vWF during the Cleaning process to an insignificant remaining amount from Factor VIII separated and the purified recombinant factor VIII stabilized with albumin (Griffith et al. (1991) Ann. Hematol 63: 166-171).

For use in the therapy of patients with haemophilia A, but also with von Willebrand syndrome is a purified factor VIII, complexed with vWF, desirable (Berntorp (1994), Haemostasis 24: 289-297). In particular, it is repeatedly emphasized that in preparations with little or no vWF content, an increased bleeding time and a low factor VIII: C half-life can be observed in vivo. A normalization of vWF in vivo is important to determine the concentration of factor VIII in the Plasma both by reducing the factor VIII elimination rate as well as by supporting the release of endogenous Factor VIII to be maintained (Lethagen et al. (1992), Ann. Hematol. 65: 253-259).

DE 3 504 385 describes the implementation of ion exchange chromatography for the purification of factor VIII / vWF complex, the factor VIII complex being bound via sulfate groups and is eluted with citrate buffer, calcium chloride and NaCl gradient. The factor VIII complex is at a concentration of 0.5 M NaCl eluted from the support.

EP 0 416 983 describes the extraction of factor VIII / vWF complex from human plasma by precipitation with a combination from barium chloride and aluminum hydroxide and subsequent Anion exchange chromatography on DEAE Fractogel.

In EP 0 411 810 the factor VIII / vWF complex is purified from cryoprecipitate by means of heparin affinity chromatography and subsequent elution of the complex with calcium chloride. A further development of this method is described in WO 93/22337. To remove contaminating proteins, such as fibrinogen and fibronectin, a glycine / NaCl precipitation is carried out after the elution with CaCl ₂ .

For the purification of the factor VIII / vWF complex it has also been proposed contaminating proteins such as fibrinogen high concentrations of amino acids, especially glycine, precipitate the factor VIII / vWF complex remaining in solution by adding a buffer containing calcium and amino acids dissociate and then factor VIII and vWF by anion exchange chromatography win separately from each other (WO 82/04395).

No. 5,356,878 describes the production of factor VIII complex, in which contaminating proteins (fibrinogen, vitamin K-dependent factors or fibronectin) are separated by precipitation with Al (OH) ₃ and PEG, factor VIII complex in the presence of glycine and NaCl chemically inactivated and then the proteins not specific to factor VIII complex are removed by gel filtration.

Hornsey et al. (Thromb. Haemost. 57: 102-105 (1987)) Factor VIII / vWF using immunoaffinity chromatography and achieved a specific activity of 45 U factor VIII / mg protein and 60 U ristocetin activity / mg protein. However the final product with 4% fibrinogen and 2% fibronectin and from the carrier detached mouse antibodies contaminated.

Mejan et al. (Thromb. Haemost. 59: 364-371 (1988)) suggested Factor VIII / vWF complex by means of immunoaffinity directly from plasma to clean. The purified complex was treated with human serum albumin stabilized and then lyophilized. With the described However, elution conditions became partial Release of the antibody from the column was observed, resulting in a Contamination of the eluate with monoclonal antibodies resulted and a second purification step to remove the antibodies required. Mejan et al. achieved one with their procedure approximately 1400-fold enrichment of the factor VIII / vWF complex with a specific factor VIII: C activity and ristocetin activity of 20 U / mg protein each, the product being all vWF multimers contained. After stabilizing the complex with 10 mg / ml A stability of 3-4 months at -20 ° C was observed for albumin. However, it is always emphasized that a special one Difficulty in cleaning the complex is that To obtain association of the proteins, since both components in the Are complex unstable.

Harrison et al. (Thromb. Res. 50: 295-304 (1988)) describes the Purification of factor VIII / vWF complex by chromatography Dextran sulfate-agarose.

EP 0 600 480 describes the purification of factor VIII / vWF complex by means of anion exchange chromatography, the eluate, containing factor VIII / vWF complex, with heparin and albumin stabilized and optionally lysine and histidine as antiproteases be added.

Commercially available factor VIII / vWF preparations show Partly no or only a small proportion of high molecular weight vWF multimers (vWF / HMW) and show in particular in dependence the infusion time in vivo a reduction in high molecular weight vWF multimers (Lethagen et al. (1992), Ann. Hematol. 65: 253-259).

The factor VIII preparations described in the prior art for the most part contain the entire vWF multimer pattern, however, they vary in the proportion of HMW-vWF and LMW-vWF and show so-called triplet structures based on proteolytic degradation of vWF multimers, especially vWF / HMW (Scott et al. (1993) Sem. Thromb. Hemost. 19: 37-47, Baillod et al. (1992) Thromb. Res. 66: 745-755, Mannucci et al. (1992) Blood 79: 3130-3137). This makes these preparations stable limited.

To stabilize the preparations, either before virus inactivation or to obtain a storage-stable preparation repeatedly stressed that the addition of a stabilizer, such as such as albumin.

All factor VIII concentrates by protein purification can be obtained from human plasma, or in contact with biological Material from mammals are also available potentially at risk, microbiological or molecular Pathogens such as Viruses, contain. For the production a safe preparation is therefore always an inactivation of pathogenic organisms necessary. Effective inactivation procedures however, can easily lead to a loss of biological activity of the factor VIII complex. So , Palmer et al. solid (Thromb. Haemost. 63: 392-402 (1990)) that heat treatment for effective virus inactivation even in the presence of a stabilizer with a Loss of activity between 17% and 30% is expected.

It is emphasized again and again that factor VIII / vWF concentrates with an intact multimer structure may have a good impact on bleeding time as they have the primary function of the vWF, platelet agglutination, and a higher one Affinity for the platelet receptors glycoprotein Ib and IIb / IIIa as low molecular weight vWF multimers (LMW-vWF) (Mannucci et al. (1987) Americ. J. Hematology 25: 55-65). It however, there is a problem that during the manufacturing process a breakdown of factor VIII concentrates in particular of the HMW-vWF molecules.

There is therefore a need for a factor VIII complex with sufficient specific activity on factor VIII: C and vWF activity that has improved stability and that even without the addition of a non-factor VIII / vWF complex-specific Stabilizer remains stable over a longer period of time.

The aim of the present invention is therefore a factor VIII / vWF complex with improved stability put.

According to the invention, the object is made available by of a factor VIII / vWF complex, which is particularly high molecular weight vWF multimers contain and are free of low molecular weight vWF molecules and proteolytic vWF degradation products.

The specific platelet agglutination gives the ratio of Ristocetin co-factor activity and vWF antigen content again. A there is therefore high specific platelet agglutination activity the specific activity of the multimers. As part of the present Invention could be used for both plasmatic vWF (p-vWF) as well as recombinant vWF (r-vWF) or factor VIII / vWF complex are shown that at a high degree of multimerization of vWF the specific platelet agglutination (RistCoF / vWF: Ag) is significantly increased compared to low molecular weight multimers.

Low molecular weight p-vWF (p-vWF / LMW) and low molecular weight r-vWF (r-vWF / LMW) have very little platelet agglutination.

This situation can be illustrated by the fact that very short vWF chain there is no stable connection of several Tile comes. In contrast, high molecular (long) vWF multimers Connect several plates together stably.

It has been shown that both high molecular weight p-vWF (p-vWF / HMW) as well as high molecular weight r-vWF (r-vWF / HMW) in a concentration-dependent manner Way to bind to platelets and a higher specific Show platelet agglutination activity as low molecular weight vWF multimers.

The stable factor VIII / vWF complex according to the invention therefore has a specific vWF platelet agglutination activity of at least 50 U / mg vWF: Ag.

In plasma, factor VIII / vWF comes in a complex in a molar ratio from about 1:50. It was found that this Ratio in plasma is necessary to provide good protection against to grant proteolytic degradation, in particular by protein C. (Vlot et al. (1995) Blood 85: 3150-3157).

According to another aspect of the present invention the factor VIII / vWF complex according to the invention has a molar ratio from factor VIII to vWF between 0.01 and 100. This means that the complex is a ratio of factor VIII molecules to vWF molecules between 1: 100 and 100: 1. Preferably the molar ratio of factor VIII to vWF is between 1:30 and 1:70, particularly preferably 1:50, with the high proportion of vWF / HMW in the complex an optimal ratio to protect against proteolytic degradation.

According to the invention, a stable factor VIII / vWF complex continues provided the high-molecular plasmatic vWF multimers with a doublet structure.

It has been found in the context of the present invention that from plasma or cryoprecipitate by a chromatographic Process purified factor VIII complex is obtained from vWF multimer molecules exist that have a doublet structure. This was particularly surprising because of plasma or cryoprecipitate purified vWF or factor VIII / vWF complex was only known with a triplet structure of the vWF multimers. These triplet structures result from proteolytic degradation of vWF multimers and indicate instability of the vWF multimers out. By Palmer et al. (Thromb. Haemost. 63: 392-402 (1990)) has described that in the production of factor VIII concentrate from heparinized plasma and subsequent Virus inactivation the normal triplet pattern changes and the intensity of the lowest molecular weight triplet band increases strongly, which indicates an increased proteolytic Degradation of the vWF multimers indicates. In contrast to that a factor VIII / vWF is available in the present invention who completely lacks this vWF degradation product and who essentially the 2 bands of the original triplet with contains the higher molecular weight.

According to a further embodiment of the invention, a stable one Factor VIII / vWF complex provided the high molecular weight recombinant vWF multimers with a singlet structure contains. It was determined by multimer analysis that the vWF multimers of recombinant vWF are only a singlet band have a high structural integrity and are free of any proteolytic vWF degradation products. The factor VIII complex according to the invention, containing high molecular weight recombinant vWF molecules with a high structural Integrity is therefore very stable and free of low molecular weight vWF multimers and vWF degradation products.

The stable factor VIII / vWF complex according to the invention is preferred free from plasma proteins, in particular from plasma proteases, and free of fibrinogen and fibronectin. Because plasma proteases and plasma proteins, in particular activated plasma proteins, such as protein C, factor IIa or factor IXa, vWF or factor VIII degrade proteolytically and the complex according to the invention is free of plasma proteins, it has increased stability and Integrity of the proteins in the complex.

The complex according to the invention has protection against proteolytic degradation increased resistance, which makes it, for example, at room temperature, for at least 48 hours, preferably for at least 6 Is stable for days, and in lyophilized form at 4 ° C or room temperature is stable in storage for more than 2 years.

The factor VIII / vWF complex according to the invention is so stable that it will be made available as a virus-safe complex can. Virus security is through procedural treatment steps of the complex for inactivating viruses or depletion guaranteed by viruses.

Heat treatment is particularly suitable for inactivating viruses in solution or solid, preferably lyophilized Condition that enveloped both lipid and non-lipid Can reliably inactivate viruses. For example, the Complex according to the invention according to EP 0 159 311 in solid, wet Condition heat treated. Other methods of virus inactivation also include treatment with detergents or chaotropic agents Substances, for example according to EP 0 519 901, WO 94/13329, DE 44 34 538 and EP 0 131 740.

According to another aspect of the invention, a stable, virus-safe Factor VIII complex concentrate, containing in particular high molecular vWF multimers with high structural integrity, made available. The high molecular vWF multimers consist preferably of a singlet or a doublet structure and are free of low molecular weight vWF multimers and proteolytic degradation products from the vWF. It has surprisingly turned out to be shown that the factor VIII complex concentrate according to the invention is so stable that treatment for Virus inactivation, as described above, stability the proteins, especially the high molecular weight vWF multimers in the Complex only insignificantly impaired and that the specific Factor VIII activity: C and vWF ristocetin activity in factor VIII complex or factor VIII / complex concentrate only reduced by a maximum of 10% during a virus inactivation step is. With previously known factor VIII complex concentrates was at a loss of virus inactivation Activity between 20 and 30%. The invention Factor VIII / vWF complex showed no changes in the neoantigen test the antigen structure after the virus inactivation step, which confirms the stability of the proteins in the complex. by virtue of The high stability of the high molecular weight vWF multimers is also a high specific platelet agglutination activity of at least 50 U / mg vWF: Ag in the factor VIII complex concentrate according to the invention guaranteed.

According to a particular embodiment, the invention contains stable, virus-safe factor VIII complex concentrate factor VIII and vwF in a molar ratio of factor VIII to vWF between 0.01 and 100, preferably between 0.05 and 1. Das Factor VIII complex concentrate is in particular free of plasma proteins, especially of plasma proteases, and free of microbiological and molecular biological pathogens.

To improve the stability of purified proteins usually stabilizers such as albumin are added. However, the specific protein is added by adding the foreign protein Purified protein activity decreased. vWF is a natural component of the factor VIII complex. It was found that by adding high molecular weight vWF multimers (vWF / HMW) to a purified factor VIII fraction from plasma or the stability of factor from recombinant cell cultures VIII is increased. It was also found that by adding vWF / HMW to a purified factor VIII complex the stability of the complex can be improved. On the addition of usually stabilizers used can therefore be dispensed with. In exceptional cases, this can also be done during extraction A protease inhibitor can be added to the intact Structure, especially the vWF / HMW.

According to the invention, a pharmaceutical composition, containing a stable factor according to the invention VIII / vWF complex or a virus-safe, stable according to the invention Factor VIII complex concentrate.

In a particular embodiment, the pharmaceutical contains Composition a physiologically acceptable carrier or buffer. The formulation of the pharmaceutical according to the invention Preparation can be carried out in a conventional manner known per se, e.g. with the help of salts and possibly amino acids, but also in the presence of surfactants. by virtue of the high stability of the complex described above can the commonly used stabilizers or protease inhibitors if necessary in the pharmaceutical composition also be dispensed with.

The stable factor VIII / vWF complex according to the invention is preferred obtained as a high-purity product, which by chromatographic Cleaning process is obtained. The chromatographic Purification is carried out in particular by ion exchange chromatography and / or affinity chromatography. For that you can et al Anion exchange materials, such as synthetic carrier materials or carbohydrate-based carriers with ligands such as DEAE, TMAE, QAE, Q or aminoalkyl groups are used, or for the affinity chromatography carrier with immobilized Substances that have a specific affinity for vWF. Suitable affinity materials include, for example Heparin.

According to a further aspect of the present invention, a method for obtaining stable factor VIII / vWF complex is therefore provided. Factor VIII / vWF complex is bound from a protein solution at a low salt concentration to an affinity carrier, preferably a heparin affinity carrier, and stable factor VIII / vWF complex is obtained at a high salt concentration. The complex is preferably bound to immobilized heparin at a salt concentration of 150 150 mM and eluted at a salt concentration of 200 200 mM and 300 300 mM. In a particularly preferred embodiment of the present invention, factor VIII / vWF complex is carried out in a CaCl ₂ -free buffer system. In this way, factor VIII / vWF complexes containing low molecular weight vWF molecules or high molecular weight vWF molecules can be selectively separated from one another and factor VIII / vWF complex containing in particular high molecular weight vWF molecules can be obtained at a higher salt concentration. Soluble mono- and divalent salts can be used for the elution. NaCl is preferably used. Calcium salts are not suitable for elution.

The method according to the invention is preferably carried out on a heparin affinity chromatography column carried out. For affinity chromatography any carrier can be bound to the heparin can be used. Proved to be well suited for example AF-Heparin Toyopearl® (a synthetic, large-pore, hydrophilic polymer based on methacrylate) (Tosohaas), Heparin EMD Fraktogel® (a synthetic, hydrophilic Polymer based on ethylene glycol, methacrylate and Dimethylacrylate) (Merck) or Heparin Sepharose Fast Flow® (containing natural dextran or Agarose derivatives) (Pharmacia).

In the method according to the invention, a buffer solution is used as the buffer system consisting of buffer substances, especially Tris / HCl, Phosphate buffer or citrate buffer, and optionally salt, which are preferably free of stabilizers, amino acids or other additives.

Affinity chromatography is preferably in a pH range from 6.0 to 8.5, preferably at a pH of 7.4 carried out.

In the method according to the invention, a protein solution containing Factor VIII / vWF complex, such as a plasma fraction, a cryoprecipitate or a cell free culture supernatant from transformed cells. The solution can also be a Enriched protein fraction from a chromatographic process his.

According to the method according to the invention for obtaining the factor VIII / vWF complex can be done in an efficient and simple manner a factor VIII / vWF complex, essentially containing high molecular weight vWF multimers. After this procedure leaves is therefore a physiologically particularly active factor VIII / vWF complex produce with good yields and in high purity. The factor VIII / vWF complex obtained in this way is particularly noteworthy by a specific activity of factor VIII: C of at least 50 U / mg factor VIII: Ag and a specific vWF platelet agglutination activity of at least 50 U / mg vWF and is especially free of low molecular weight vWF multimers and vWF degradation products.

According to another aspect of the present invention, a Process for the production of stable factor VIII / vWF complex for Provided in the factor VIII / vWF complex from one impure protein solution bound to an anion exchanger and contaminating plasma proteins at a salt concentration of ≤ 200 mM can be selectively eluted in the presence of calcium salt. Then factor VIII / vWF complex from the anion exchanger obtained with a salt concentration between ≤ 200 and ≤ 400 mM. It becomes a factor VIII / vWF complex, essentially containing high molecular weight vWF multimers.

To carry out the method according to the invention can be considered impure Protein solution containing factor VIII / vWF complex, e.g. a plasma fraction, a cryoprecipitate or a cell-free one Culture supernatant from transformed cells can be used.

The contaminating proteins removed by the calcium salts are, in particular, plasma proteins, including factors dependent on vitamin K, such as, for example, factor II, factor IX, protein C, protein S, plasma proteases such as plasminogen, fibronectin or fibrinogen. The non-specific proteins are removed in particular with CaCl ₂ as calcium salt in the eluent in a concentration between 1 mM and 15 mM, preferably of 10 mM.

In the context of the present invention, it was found that the previously used methods of aluminum hydroxide treatment for the separation of vitamin K-dependent proteins, fibrinogen or fibronectin is not sufficient to complete these proteins to remove. By elution in the presence of calcium chloride however, it was ensured that these plasma proteins to be essentially eliminated and a plasma protein-free Factor VIII / vWF complex is obtained.

Anion exchange chromatography is preferably carried out in one pH range from 6.0 to 8.5, preferably at a pH of 7.4.

Elution of the anion exchange chromatography on the Anion exchanger bound factor VIII / vWF complex takes place preferably by increasing the salt concentration.

An anion exchanger is preferably used as the anion exchanger of the quaternary amino type, in particular a fractogel with tent structure and in particular EMD-TMAE Fractogel is used.

Preferably, factor VIII / vWF complex is added to the anion exchanger bound at a salt concentration of ≤ 200 mM and at a salt concentration of ≥ 270 mM, preferably at ≥ 350 mM, Factor VIII / vWF complex, essentially containing vWF / HMW, eluted. Soluble mono- and divalent salts can be used as salts, NaCl is preferred.

This is preferably done by anion exchange chromatography purified factor VIII complex continues by affinity chromatography, preferably on immobilized heparin, in a buffer solution consisting of buffer substances and optionally Salt, chromatographically cleaned.

In a special embodiment of the method according to the invention first becomes a factor obtained from cryoprecipitate VIII / vWF complex-containing fraction to an anion exchanger bound and after separation of the accompanying proteins, in particular the plasma proteins, in enriched form factor VIII / vWF complex, containing essentially high molecular weight vWF multimers, eluted. In a further cleaning step, the factor VIII / vWF complex-containing eluate with an affinity carrier with covalently bound heparin in contact with the complex binds to the carrier. After removing foreign substances and foreign proteins by a suitable eluent Factor VIII complex from the affinity carrier using a single or divalent salt, preferably NaCl, in a buffer system eluted.

In a further special embodiment, this is carried out the method with obtained from recombinant cells Factor VIII / vWF complex. A cell-free culture supernatant can do this of cells that co-express factor VIII and vWF, or of cocultivated cells with factor VIII on the one hand and transformed with vWF on the other hand can be used.

By the method according to the invention for obtaining a highly pure stable factor VIII complex can be simple and efficient way a high purity factor VIII / vWF complex that is free of antibodies, free of plasma proteins, physiological active and free of microbiological and molecular biological Is pathogenic.

The factor obtained from plasma or from recombinant cells VIII / vWF complex contains according to the present invention in particular high molecular weight vWF multimers, and is free of low molecular weight vWF molecules and vWF degradation products. Becomes the factor VIII / vWF complex obtained from plasma or cryoprecipitate, see above vWF / HMW consist in particular of doublet structures with high Stabiliät. Factor VIII / vWF complex obtained from recombinant cells contains high molecular weight vWF molecules with a singlet structure, high stability and structural integrity.

By means of defined chromatography steps, it is therefore in accordance with of the present invention possible, FVIII / vWF, free from others Coagulation factors and other plasma proteases. This affects in particular the purity and stability of the Preparation cheap. The factor obtained according to the invention VIll / vWF complex is therefore in a particularly pure form. The purity of the complex is preferably at least 90%, especially preferably 95%.

The fact that the factor VIII / vWF complex obtained by the high content of high molecular weight vWF multimers and the absence of low molecular weight multimers, vWF degradation products, Foreign proteins, e.g. Plasma proteins, is particularly stable, it is not absolutely necessary to use stabilizers for cleaning Add product. This makes the specific activity of the pure product e.g. in the formulation of pharmaceutical Composition, not degraded and it is avoided that if necessary, impurities by the addition of foreign proteins or infectious particles introduced into the product become.

In a further aspect of the invention, a method for Preparation of a stable factor VIII / vWF complex is available posed. It is a chromatographic Process purified factor VIII or factor VIII complex one added purified high molecular fraction of vWF molecules, creating a factor VIII / vWF complex with a molar ratio from factor VIII to vWF / HMW between 0.01 (corresponds to 1 factor VIII: 100 vWF) and 100 (corresponds to 100 factor VIII: 1 vWF), preferably between 0.03 (1:30) and 0.07 (1:70), particularly preferred of 0.05 (1:50) is obtained.

The factor purified using a chromatographic process VIII or factor VIII complex can be from a plasma fraction, a cryoprecipitate or from a cell-free cell culture supernatant derived from transformed cells.

The process for the preparation of the stable complex High molecular weight fraction of vWF molecules can be used from plasma, a plasma fraction, a cryoprecipitate or a cell-free culture supernatant from transformed cells come. The purified high molecular fraction of vWF molecules preferably contains a specific platelet agglutination activity of at least 50 U / mg vWF: Ag, particularly preferred of at least 80 U / mg vWF: Ag.

A purified is used to produce the stable factor VIII complex Fraction containing factor VIII or factor VIII / vWF complex, with a purified high molecular fraction of vWF molecules mixed in a desired molar ratio. To the content of vWF, factor VIII, factor VII activity and specific vWF activity determined and by adding the appropriate amount of vWF / HMW that desired mixing ratio set. Preferably done the mixture in such a way that a factor VIII / vWF complex is formed, which has a molar ratio of factor VIII to vWF between 1: 100 and 100: 1, preferably 1:50.

According to the present invention, however, a factor can also be used VIII / vWF complex are made up of a certain ratio from specific factor VIII: C activity to specific vWF platelet agglutination activity. Particularly preferred is a complex that is a ratio of the specific Has activities of 1: 1. The desired mixing ratio to manufacture is in the general state of knowledge of one Professional.

The one provided according to the present invention stable virus-safe factor VIII complex as well as the factor VIII complex concentrate can be used both for the treatment of hemophilia A as well as for the treatment of von Willebrand syndrome become. Since the factor VIII complex preparation according to the invention contains essentially high molecular weight vWF molecules it is particularly suitable for the treatment of vWD type II.

Due to the high proportion of high molecular vWF multimers the complex according to the invention also has very good pharmokinetics, because the vWF / HWM have a higher specific platelet agglutination and have stability in vitro and in vivo and both Stabilize factor VIII in vitro and in vivo. Through the Stability and structural integrity of the vWF multimers in the complex is an improved half life of the vWF and in particular also given by factor VIII, which may result in the intervals an administration of the pharmaceutical according to the invention Preparation can be reduced. This is especially the Occurrence of inhibitory antibodies against factor VIII Haemophilia A patients, e.g. through frequent doses of factor VIII concentrates prevented.

The invention is based on the following examples and the Drawing figures explained in more detail, but not on this is limited.

Show it:

Figure 1 : SDS-PAGE analysis of the vWF multimer pattern of the individual fractions before and after anion exchange chromatography.

Figure 2 : Detection of factor II in individual fractions before and after anion exchange chromatography and after removal of factor II by calcium chloride elution.

Figure 3 : Detection of protein S in individual fractions before and after anion exchange chromatography and after removal of protein S by calcium chloride elution.

Figure 4 : Detection of factor IX in individual fractions before and after anion exchange chromatography and removal of factor IX by calcium chloride elution.

Figure 5 : Detection of plasminogen in individual fractions before and after anion exchange chromatography and removal of plasminogen by previous lysine-Sepharose.

Figure 6 : SDS-PAGE analysis of the vWF multimer pattern of the individual fractions before and after heparin affinity chromatography.

Figure 7 : Multimer analysis of p-vWF and r-vWF before and after heparin affinity chromatography.

Figure 8 : Comparison of the binding of r-vWF / HMW and p-vWF / HMW to platelets and graphic representation of the added amount of vWF and platelet-bound amount of vWF.

Figure 9 : Binding of p-vWF / HMW and r-vWF / HMW to platelets and multimer analysis.

Example 1: Purification of factor VIII / vWF complex by anion exchange chromatography

10 g cryoprecipitate were dissolved with 70 ml Na acetate, pH 7.0, 160 mM NaCl and 50 U / ml heparin at 30 ° C for 10 min and incubated at room temperature for a further 30 min until completely dissolved. The solution was cooled to 15 ° C and centrifuged until undissolved components were removed and the supernatant was treated with aluminum hydroxide gel. The solution was bound to a Fractogel-EMD-TMAE anion exchanger and the anion exchanger was washed with 180 mM NaCl and 200 mM NaCl to remove foreign proteins. vWF and FVIII were then eluted as a complex with 400 mM NaCl. The factor VIII: C and vWF: RistoCoF activity of the starting material and the individual fractions were determined and are summarized in Table 1. Factor VIII: C and vWF: RistoCoF activity of the aluminum hydroxide supernatant and the fractions of the anion exchange chromatography sample volume vWF: RistCoF FVIII: C (Ml) (MU / ml) (MU / ml) Aluminum supernatant 147 1060 2970 180 mM eluate 254 - - 200 mM eluate 210 - - 400 mM eluate 132 1590 2890

By anion exchange chromatography factor VIII / vWF complex with increased vWF: RistoCoF activity become. To examine the vWF polymer pattern, the individual Anion exchange chromatography fractions on SDS-PAGE (Laemmli (1970), Nature 227: 680-685) analyzed (Figure 1 B). The polymer pattern of the vWF in the purified factor VIII / vWF complex shows an identical band pattern and thus the same vWF polymer composition as in cryoprecipitate. The cleaning therefore did not lead to a proteolytic degradation of high molecular weight vWF multimers in a complex.

Example 2: Removal of vitamin K-dependent proteins and recovery of high purity factor VIII / vWF complex

The aim of the investigations was to obtain an FVIII / vWF complex, free from proteases and other coagulation factors. As described in Example 1, dissolved cryoprecipitate was treated with aluminum hydroxide and then purified by anion exchange chromatography. To remove non-factor VIII / vWF complex-specific proteins, 10 mM CaCl _{2 were} added during the elution with 180 mM NaCl. The factor VIII: C and vWF: RistoCoF activity of the starting material and the individual fractions were determined and are summarized in Table 2. Factor VIII: C and vWF: RistoCoF activity of the starting material and the individual fractions of the anion exchange chromatography sample volume vWF: RistCoF FVIII: C (Ml) (MU / ml) (MU / ml) Aluminum supernatant 146 1590 4250 180 mM eluate / 10 mM CaCl ₂ 195 - - 200 mM eluate 127 - - 400 mM eluate 83 2120 5140

The eluates were SDS-PAGE on their vWF multimer pattern examined (Figure 1A).

From the multimer analysis it can be seen that the 400 mM eluate contains the high molecular weight vWF multimers and that there is no loss starting from the cryoprecipitate, in particular in high molecular weight vWF multimers. By adding CaCl ₂ ions, low molecular weight vWF is separated off and a factor VIII complex containing a higher proportion of high molecular weight vWF molecules is obtained (FIG. 1A). This shows that the purification process avoids proteolytic degradation of the high molecular weight vWF multimers and, by adding CaCl ₂ ions, selectively low molecular weight vWF molecules, such as dimers or tetramers, can be removed, whereby a factor VIII complex in the essentially containing high molecular weight vWF multimers.

The individual cleaning stages, containing factor VIII / vWF complex, have been linked to the presence of vitamin K-dependent proteins analyzed before and during anion exchange chromatography. For this purpose, individual cleaning stages and the individual Fractions separated on an SDS-PAGE, the proteins on one Membrane transferred and the vitamin by Western blot analysis Detected K-dependent proteins.

a. Evidence of factor II in the individual cleaning stages

For the detection of factor II in the individual fractions polyclonal serum against factor II (Assera factor II, Stago) as 1. Antibodies used and conjugated with alkaline phosphatase polyclonal goat anti-rabbit IgG-HRP conjugate (Fa. Bio-Rad) as 2nd antibody and subsequent chromogenic test detected.

It can be seen from FIG. 2 that cryoprecipitate contains factor II. Despite previous aluminum hydroxide precipitation, this is eluted as an impurity at 400 mM NaCl (together with FVIII / vWF) from the anion exchanger (lane E). However, factor II can be selectively eluted by 10 mM CaCl ₂ (lane F), and vWF / FVIII can be obtained free of factor II (lane G) on subsequent elution with 400 mM NaCl.

b. Detection of Protein S in the individual cleaning stages

Polyclonal was used to detect Protein S in the purification stages Rabbit anti-protein S serum (Assera Protein S, Stago) was used as the 1st antibody and with goat-anti-rabbit-IgG-HRP conjugate (Bio-Rad) as 2nd antibody and subsequent chromogenic test detected.

FIG. 3 shows the detection of protein S in individual purification stages before and after anion exchange chromatography and after removal of Protein S by calcium chloride elution.

From FIG. 3 it can be seen that protein S from cryoprecipitate, despite previous aluminum hydroxide precipitation, eluted as an impurity at 400 mM NaCl (together with FVIII / vWF) from the anion exchanger (lane E). Protein S can, however, be selectively eluted by 10 mM CaCl ₂ (lane F), and FVIII / vWF can be obtained free of protein S on subsequent elution with 400 mM NaCl (lane G).

c. Detection of factor IX in the individual cleaning stages

To detect factor IX in the cleaning stages, polyclonal Rabbit anti factor IX serum (Assera factor XI, Stago) was used as the 1st antibody and with goat-anti-rabbit-IgG-HRP conjugate (Bio-Rad) as 2nd antibody and subsequent chromogenic test detected.

FIG. 4 shows the detection of factor IX in individual cleaning stages before and after anion exchange chromatography and selective removal of factor IX by calcium chloride.

It can be seen from FIG. 4 that factor IX elutes from the anion exchanger as an impurity at 400 mM NaCl (together with factor VIII complex) despite previous aluminum hydroxide precipitation. However, factor IX is selectively eluted by adding 10 mM CaCl ₂ (lane D) and FVIII / vWF is obtained free of factor IX (lane E) on subsequent elution with 400 mM NaCl.

Example 3: Removal of plasma proteases and recovery of high purity Factor VIII / vWF complex

The aim of the investigations was to obtain a vWF / FVIII preparation, free of proteases and other coagulation factors. An essential one The protease constitutes contamination of the cryoprecipitate Plasminogen. This is also found in the FVIII / vWF eluate (400 mM eluate) again.

To remove plasminogen, cryoprecipitate was used as above described, dissolved and treated with aluminum hydroxide gel. The aluminum supernatant was then passed through a lysine-Sepharose gel filtered, and directly on the anion exchanger (Fractogel EMD TMAE) applied. FVIII / vWF was, as described, eluted from the anion exchanger with 400 mM NaCl. The eluate before and after anion exchange chromatography were on Plasminogen analyzed by Western blot (Figure 5). To do this the proteins are separated by gel electrophoresis using SDS-PAGE, blotted onto a membrane and plasminogen with a polyclonal rabbit anti-plasminogen serum (Stago) as 1st Antibody and subsequent chromogenic test detected.

From the results it can be seen that by filtration Lysine-Sepharose selectively protease plasminogen from vWF / FVIII is separated.

Example 4: (currently in the opinion of the applicant of the best way to carry out the invention) Heparin affinity chromatography of FVIII / vWF complex

Fractogel AF EMD heparin was used for heparin affinity chromatography. FVIII / vWF, which was purified by anion exchange chromatography according to Example 2 (400 mM eluate), served as the starting material. To purify FVIII / vWF via affinity chromatography, 27 ml of the 400 mM NaCl fractogel eluate were diluted 4-fold with 81 ml of Tris-HCl buffer (pH 7.4) and applied to the heparin affinity column. The column was washed first with 100 mM NaCl and then with 160 mM NaCl to remove non-specifically bound proteins. The factor VIII / vWF complex was then obtained by eluting the heparin column with 300 mM NaCl. The factor VIII: C and vWF: RistoCoF activity as well as the vWF: Ag content in the starting material and the individual fractions of the anion exchange chromatography and the heparin affinity chromatography were determined and are summarized in Tables 4 A and 4 B. Factor VIII: C and vWF: RistoCoF activity of the starting material and the individual fractions before and after anion exchange chromatography sample volume vWF: Ag vWF: RistCoF FVIII: C (Ml) (Ug / ml) (MU / ml) (MU / ml) Aluminum supernatant 75 55 1700 5260 180 mM eluate / 10 mM CaCl ₂ 64 21 43 - 200 mM eluate 33 1 - - 400 mM eluate 29 137 4250 9500 Factor VIII: C and vWF: RistoCoF activity of the starting material and the individual fractions before and after heparin affinity chromatography sample volume vWF: Ag vWF: RistCoF FVIII: C (Ml) (Ug / ml) (MU / ml) (MU / ml) starting material 104 42 850 2630 160 mM eluate 42 11 43 650 300 mM eluate 28 92 2550 5840

The eluates of heparin affinity chromatography were based on vWF polymer composition examined (Figure 6).

It can be seen from FIG. 6 that the high molecular weight fraction of the vWF is obtained in the 300 mM NaCl fraction. This faction also has the highest factor VIII: C and vWF ristocetin cofactor activity (Table 4 B).

From the sum of the results it can be clearly seen that the combination of anion exchange chromatography and heparin affinity chromatography both vWF, FVIII, and their complex Isolate from cryoprecipitate and have cleaned. In particular with heparin affinity chromatography it is possible vWF low molecular weight multimers and degradation products from vWF Separate cryoprecipitate.

Example 5: Determination of the specific ristocetin cofactor activity of purified vWF or vWF complex

By means of chromatographic methods, plasmatic vWF (p-vWF) from human cryoprecipitate and recombinant vWF (rvWF) the fermentation supernatant of recombinant CHO cells is isolated and cleaned according to Example 2. By heparin affinity chromatography and elution with different salt concentrations Fractions with different degrees of polymerization on vWF isolated (according to Example 4). Overall, p-vWF and r-vWF fractions with low-molecular-weight vWF (vWF / LMW) at 120 mM NaCl, with medium molecular weight (vWF / MMW) at 230 mM NaCl and high molecular weight (vWF / HMW) at 300 mM NaCl. These fractions were checked for their content of vWF: Ag by ELISA (Asserachrom vWF®, Boehringer Mannheim), on ristocetin cofactor activity (vWF reagent, Behringwerke), their multimer structure examined by SDS-PAGE and their platelet binding.

FIG. 7 shows the vWF polymer analysis of plasmatic vWF and recombinant vWF.

It is particularly striking that before cleaning plasma vWF in the cryoprecipitate the vWF dimers, tetramers or multimers exist as triplet structures. These triplet structures are degradation products of vWF multimers and are in the plasma occurring protease attributed. After cleaning are especially with the vWF-MMW and vWF-HMW fractions only Recognize multimers with duplet structures. By the chromatographic Procedures are so vWF multimers with changed Composition and structure compared to those found in plasma vWF molecules obtained, indicating a depletion of Proteases and low molecular weight vWF degradation products is.

Compared to plasmatic vWF multimers, recombinant vWF clearly the presence of only one singlet band in the vWF multimers to recognize. The recombinant vWF multimer molecules have high structural integrity and contain none proteolytic degradation products, compared to those from the literature known vWF triplet structures of the plasmatic vWF.

Table 5 and Table 6 show the specific ristocetin cofactor activity (RistCoF / vWF: Ag) for p-vWF and r-vW. Specific ristocetin cofactor activity for different p-vWF fractions sample Specific RistCoF activity (mU RistCoF / vWF: Ag) p-vWF / LMW 3 p-vWF / MMW 10 p-vWF / HMW 56 Specific ristocetin cofactor activity for different r-vWF fractions sample Specific RistCoF activity (mU RistCoF / vWF: Ag) r-vWF / LMW 1 r-vWF / MMW 6 r-vWF / HMW 41

Example 6: Binding of p-vWF and r-vWF to Platelets

The binding of p-vWF and r-vWF examined on platelets. p-vWF / HMW and r-vWF / HMW, respectively incubated with constant concentration of platelets and ristocetin. The platelets were then centrifuged separated (platelet sediment, bound vWF) and a supernatant (unbound vWF) received. In the source material and in The supernatant was vWF: Ag and the platelet-bound amount of vWF certainly. Identical incubations without ristocetin were used as controls carried out. These did not result in vWF platelet bindings. The ratio of vWF concentration in the incubation batch and Platelet-bound vWF is shown in Figure 8 and shows in direct comparison of the binding behavior of p-vWF / HMW and r-vWF / HMW. Following the incubations, both the Supernatants (unbound vWF) and vWF in platelet sediment (bound vWF) examined for multimer composition. The results of the multimer analysis are summarized in FIG. 9.

Example 7: Determination of the stability of purified factor VIII / vWF complex

By anion exchange or heparin affinity chromatography Fractions obtained were determined on the stability of the vWF multimers as well as factor VIII activity were examined. For this, the in fractions obtained in the individual purification steps -20 ° C, 4 ° C and room temperature for a period of up to 60 days stored and after 0, 1, 3, 5, 10, 15, 20, 30 and 60 days Samples from a vWF multimer analysis, a factor VIII: C and vWF ristocetin cofactor activity determined. The Eluates of anion exchange chromatography or heparin affinity chromatography, in which the plasma protease due to lysine-Sepharose or by calcium chloride elution the vitamin K-dependent Factors had been removed selectively greatest stability. The vWF multimer pattern was in these samples unchanged even after 30 days, while in the samples none Lysine-Sepharose chromatography or calcium chloride elution had an appearance depending on the length of time of proteolytic vWF degradation products. In particular is therefore responsible for maintaining the stability of the high molecular weight vWF multimers the removal of in the starting material existing plasma proteins are necessary, as these have storage stability strongly influence or reduce.

Example 8: Increased stability of the purified factor VIII complex by adding purified high molecular weight vWF multimers

Different by chromatographic purification steps obtained factor VIII or factor VIII / vWF complex-containing Fractions were different amounts of purified p-vWF / HMW or r-vWF / HMW added, the mixtures at 4 ° C and room temperature incubated for up to 40 days and the vWF multimer composition and factor VIII: C and vWF ristocetin cofactor activity after 0, 1, 5, 10, 20, 25, 30, 35 and 40 days determined. The stability of the vWF multimers as well the specific ristocetin cofactor activity was in eluates, in which plasma proteins by previous chromatography, especially plasma proteases, had been removed best. By adding a vWF / HMW-containing fraction to the individual Fractions containing factor VIII or factor VIII / vWF or to the starting material from cryoprecipitate could in particular in the fractions according to Example 7 have a lower stability had an improvement in stability can be achieved. Dependent from the addition of the amount of high molecular weight vWF multimers could prevent proteolytic breakdown products from occurring as well a reduction in specific activity on factor VIII and vWF activity may be delayed.

Example 9: Virus inactivation of purified factor VIII / vWF complex or purified factor VIII / vWF complex after adding high molecular weight vWF multimers and determination of factor VIII: C and vWF activity RistoCoF

Individual fractions of the chromatographic purification steps, as well as fractions, to which purified high-molecular vWF multimers or albumin were added, a virus inactivation procedure subjected. To do this, the samples were for 10 hours heated to 60 ° C and then again at 80 ° C for 1 hour continuing incubation. Then a vWF multimer analysis was carried out and a determination of factor VIII: C and specific activity vWF platelet agglutination activity performed. It it was found that in particular samples obtained after heparin affinity chromatography a particularly high proportion of high molecular weight vWF multimers and no low molecular weight vWF degradation products contain, as well as a high specific ristocetin cofactor activity had the least loss of activity Factor VIII and vWF showed. Even with samples, which additionally a certain amount of purified high molecular weight vWF multimers was added, the loss of activity after Inactivation procedure maximum 10%. In the samples to which albumin had been added, the specific activity decreased when added of the stabilizer and then again after the inactivation process. This showed that the presence only vWF / HMW or by adding high molecular weight vWF multimers factor stability of the proteins VIII / vWF complex can be increased significantly without the specific activity is significantly reduced.

Claims (43)

1. A stable factor VIII/vWF-complex particularly comprising high-molecular vWF multimers and being free from low-molecular vWF molecules and from proteolytic vWF degradation products.
2. A stable factor VIII/vWF-complex according to claim 1, characterized in that it exhibits a specific platelet agglutination activity of at least 50 U/mg vWF:Ag.
3. A stable factor VIII/vWF-complex according to claim 1 or 2, characterized in that it has a molar ratio of factor VIII to vWF of between 0.01 and 100.
4. A stable factor VIII/vWF-complex according to claim 3, characterized in that the molar ratio of factor VIII to vWF is between 0.05 and 1.
5. A stable factor VIII/vWF-complex according to any one of claims 1 to 4, characterized in that it comprises high-molecular plasmatic vWF multimers having doublet structure.
6. A stable factor VIII/vWF-complex according to any one of claims 1 to 5, characterized in that it comprises high-molecular recombinant vWF multimers having singlet structure.
7. A stable factor VIII/vWF-complex according to any one of claims 1 to 6, characterized in that the high-molecular vWF molecules have high structural integrity.
8. A stable factor VIII/vWF-complex according to any one of claims 1 to 7, characterized in that it is free from plasma proteins, in particular plasma proteases, and free from fibrinogen and fibronectin.
9. A stable factor VIII/vWF-complex according to any one of claims 1 to 8, characterized in that it is storage-stable in solution.
10. A stable factor VIII/vWF-complex according to any one of claims 1 to 9, characterized in that it has been treated for inactivation or depletion of viruses.
11. A stable, virus-safe factor VIII/vWF-complex concentrate, particularly comprising high molecular vWF multimers of high structural integrity, the vWF multimers being comprised of a singlet or doublet structure and being free from proteolytic degradation products of vWF.
12. A stable, virus-safe factor VIII/vWF-complex concentrate according to claim 11, characterized in that it has a specific platelet agglutination activity of at least 50 U/mg vWF:Ag.
13. A stable, virus-safe factor VIII/vWF-complex concentrate according to any one of claims 11 to 12, characterized in that it has a molar ratio of factor VIII to vWF of between 0.01 and 100.
14. A stable, virus-safe factor VIII/vWF-complex concentrate according to claim 13, characterized in that the molar ratio of factor VIII to vWF is between 0.05 and 1.
15. A stable, virus-safe factor VIII/vWF-complex concentrate according to any one of claims 11 to 14, characterized in that it is free from plasma proteins, in particular plasma proteases, and free from microbiological and molecular-biological pathogens.
16. A pharmaceutical composition containing a stable factor VIII/vWF-complex according to any one of claims 1 to 15.
17. A pharmaceutical composition according to claim 16, characterized in that it comprises a physiologically acceptable carrier.
18. The use of a stable factor VIII/vWF-complex according to any one of claims 1 to 15 for producing an agent for treating hemophilia A and/or various forms of von Willebrand syndrome.
19. A method of recovering stable factor VIII/vWF- complex, characterized in that factor VIII/vWF-complex from a protein solution is bound to a heparin affinity carrier and factor VIII/vWF-complex is recovered at a salt concentration of between ≥ 200 and ≤ 300 mM.
20. A method according to claim 19, characterized in that a plasma fraction or a cryoprecipitate comprising factor VIII/vWF-complex is used as the protein solution.
21. A method according to claim 19, characterized in that a culture supernatant from transformed cells which is free from cells and comprises factor VIII/vWF-complex is used as the protein solution.
22. A method according to claim 19, characterized in that an enriched protein fraction is used as the protein solution.
23. A method according to any one of claims 19 to 22, characterized in that a carrier with heparin bound thereto, preferably AF-Heparin Toyopearl® (Tosohaas), Heparin EMD-Fraktogel® or Heparin-Sepharose Fast Flow®, is used as the heparin affinity carrier.
24. A method according to any one of claims 19 to 23, characterized in that a buffer solution comprised of buffer substances, preferably Tris/HCl buffer, phosphate buffer or citrated buffer and, optionally, salt is used as the buffer system for the affinity chromatography.
25. A method according to any one of claims 19 to 24, characterized in that the affinity chromatography is effected in a pH range of from 6.0 to 8.5, preferably at a pH of 7.4.
26. A method according to any one of claims 19 to 25, characterized in that NaCl is used as salt.
27. A method according to any one of claims 19 to 26, characterized in that a factor VIII/vWF-complex is obtained with a specific activity of factor VIII:C of at least 50 U/mg protein and a specific platelet agglutination activity of at least 50 U/mg vWF.
28. A method according to any one of claims 19 to 27, characterized in that a factor VIII/vWF-complex-containing fraction, particularly comprising high-molecular vWF multimers, is obtained which is free from low-molecular vWF multimers and from vWF degradation products.
29. A method of recovering stable factor VIII/vWF-complex, wherein factor VIII/vWF-complex from a contaminated protein solution is bound to an anion exchanger, characterized in that contaminating plasma proteins are selectively eluted with CaCl₂ at a salt concentration of ≤ 200 mM, and subsequently factor VIII/vWF-complex is obtained from the anion exchanger with a salt concentration of between ≥ 200 and ≤ 400 mM.
30. A method according to claim 29, characterized in that the contaminating proteins are plasma proteins.
31. A method according to claim 30, characterized in that the plasma proteins particularly are vitamin K-dependent factors, plasma proteases, fibronectin or fibrinogen.
32. A method according to any one of claims 29 to 31, characterized in that the CaCl₂ is used in the eluting agent at a concentration of between 1 mM and 15 mM, preferably 10 mM.
33. A method according to any one of claims 29 to 32, characterized in that the elution is effected in a pH range of from 6.0 to 8.5, preferably at a pH of 7.4.
34. A method according to any one of claims 29 to 33, characterized in that NaCl is used as the salt.
35. A method according to any one of claims 29 to 34, characterized in that a factor VIII/vWF-complex particularly comprising high-molecular vWF multimers is obtained which is free from low-molecular vWF molecules and from vWF degradation products.
36. A method according to any one of claims 29 to 35, characterized in that the fraction comprising the recovered factor VIII/vWF-complex is subjected to a further chromatographic step, preferably to an affinity chromatography.
37. A method according to claim 36, characterized in that a heparin chromatography according to any one of claims 19 to 27 is used as the affinity chromatography.
38. A method of producing a stable factor VIII/vWF complex, characterized in that a purified high- molecular fraction of vWF molecules is admixed to a factor VIII or factor VIII/vWF-complex which has been purified via a chromatographic method, a factor VIII/vWF-complex having a molar ratio of factor VIII to vWF of between 0.01 and 100, preferably of between 0.05 and 1, being obtained thereby.
39. A method according to claim 38, characterized in that the purified factor VIII or factor VIII/vWF-complex is recovered from a plasma fraction.
40. A method according to claim 38, characterized in that the purified factor VIII or factor VIII/vWF-complex is recovered from a cell culture supernatant from transformed cells which is free from cells.
41. A method according to claim 38, characterized in that the purified high-molecular fraction of vWF molecules is comprised of plasmatic vWF.
42. A method according to claim 38, characterized in that the purified high-molecular fraction of vWF molecules is comprised of recombinant vWF.
43. A method according to any one of claims 38 to 42, characterized in that a high-molecular fraction of vWF molecules having a specific platelet agglutination activity of at least 50 U/mg vWF:Ag is recovered.

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